A New Tandem-Affinity Tag for Two-Step Protein Purification under Fully Denaturing Conditions
Background: Preservation of posttranslational modifications during purification is crucial for successful mass spectrometric analyses of protein modifications. Current tandem-affinity purification strategies require native conditions and are therefore susceptible to loss of posttranslational modifications during cell lysis and purification because modifying as well as de-modifying enzymes remain active under these conditions. Technology: University of California, Irvine researchers have developed a novel tandem-affinity tag that is compatible with two-step purification under fully denaturing conditions such as 8 M urea or 6 M guanidinium. This novel tag serves as a biotinylation signal in vivo. Tagged proteins are efficiently biotinylated in vivo in both yeast and mammalian cells at a specific lysine residue present in the tag. The biotinylated protein can then be sequentially purified by Ni2+-chelate chromatography and binding to streptavidin resins. Application: Taken together, this novel tag and its derivatives are useful tools for proteomic studies using mass spectrometry. The tag allows tandem affinity purification under fully denaturing conditions and thus combines a high degree of purification with preservation of protein modifications in the in vivo status. In addition, the procedure is compatible with in vivo cross-linking, which allows identification of transiently associated proteins, and can thus be an important tool to probe and understand the dynamics of the proteome.Type of Offer: Licensing
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